Glutathione transferases (GSTs) are a multi-gene enzyme family which through catalyzing a number of distinct glutathione dependent reactions play critical roles in providing protection against electrophiles and products of oxidative stress. Multiple cytosolic and membrane-bound GST isoenzymes with divergent catalytic and non-catalytic binding properties are found in all eukaryotic species. The mammalian cytosolic GSTs are made up of Alpha (A), Mu (M), Omega (O), Pi (P), Sigma (S), Theta (T) and Zeta (Z) families (Strange et al., 2001). The most recently discovered class of cytosolic GSTs, the Omega class (GSTO1 and GSTO2), are characterised by a unique N-terminal extension and a cysteine residue in the active site, which is distinct from the tyrosine and serine residues associated with other GST classes. GSTO1 (Board et al., 2000) exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activites characteristic of glutaredoxins and which are not associated with other GSTs. The polypeptide consists of 241 amino acids with a predicted MW of 27.5 kDa but migrates at approximately 56 kDa suggesting it forms dimers under native conditions (Board et al., 2000). The structure of recombinant GSTO1 has been solved at 2 Angstrom resolution (NCBI Protein Database: 1EEM; Board et al., 2000). Expression of GSTO1 is abundant in a wide range of normal tissues including liver, macrophages, glial cells and endocrine cells, as well as myoepithelial cells of the breasts, neuroendocrine cells of the colon, fetal myocytes, hepatocytes, biliary epithelium, ductal epithelium of the pancreas, Hofbauer cells of the placenta and follicular and C-cells of the thyroid (Yin et al., 2001). This widespread expression and conserved sequence suggests that GSTO1 may have a significant house-keeping role and biological functions distinct from other GSTs.
The literature contains numerous reports on the role of GSTs in various stages of disease progression and treatment. Whereas the role of GSTs is largely beneficial in deactivating and detoxifying potentially dangerous chemicals, it appears that sometimes they have a detrimental effect in the body. For instance, over-expression has been linked with various forms of cancer, for example GSTO1 may be up-regulated in both colorectal (Liu et al., 2007) and pancreatic cancer (Chen et al., 2009). Over-expression of GSTO1 is also correlated with the onset of drug resistance of cancer cells. This may be the result of an association with the activation of survival pathways (Akt and ERK1/2) and inhibition of apoptotic pathways such as JNK1 and protection against cisplatin induced apoptosis (Piaggi et al., 2010).
Genetic variation in GSTs has been reported to represent a risk factor for a variety of diseases including many forms of cancer. A single nucleotide polymorphism (SNP) at base 419 (419C>A) of GSTO1 results in an alanine to aspartate substitution in amino acid 140 (A140D). Tanaka-Kagawa et al., (2003) functionally characterised recombinant GSTO1 Ala140Asp variants and discovered that enzyme activity decreased from that of WT (Ala/Ala) for particular substrates. This change in activity is a likely contributor to this SNPs role in disease. Polymorphisms in GSTO1 affecting the enzymes ability to metabolise inorganic arsenic have also been found (Chung et al., 2011; Agusa et al., 2008), leading to differences in individuals susceptibility to arsenic toxicity. The GSTO1 A140D polymorphism could play an important role as a risk factor for the development of heptacellular carcinoma, cholangiocarcinoma and breast cancer (Marahatta et al., 2006). The presence of WT (Ala/Ala) is more likely amongst cases of advanced stage breast cancer (Purisa et al., 2008; Chariyalertsak et al., 2009). The GSTO1 A140D polymorphism has also been associated with the risk of acute lymphoblastic leukaemia (ALL) in children and may also be involved in development of the disease (Pongstaporn et al., 2009). A role in chronic obtrusive pulmonary disease (COPD) has also been proposed (Harju et al., 2007).
Studies also suggest that GSTO1 is a risk indicator for Alzheimer's disease (AD) and Parkinson's disease (PD). Li et al., (2003) reported a difference in the gene expression of GSTO1 between AD patients and controls and that the single polymorphism rs4925 (equivalent to the Ala140Asp mutation) was linked to later age-at-onset (AAO) of both AD and PD. Kolsch et al., (2004) also found that GSTO1 polymorphisms were associated with an earlier AAO and increased the risk of vascular dementia and stroke. Although these contrasting findings could suggest that the SNP is not the causal factor in AAO, an association is present and warrants further investigation into its use as a marker. A recent study also supports a role for the GSTO1 Ala140Asp SNP in sporadic AD (Capurso et al., 2010) which is the most common form of AD. Wahner et al., (2007) found a 32% risk reduction for PD among subjects carrying one or more GSTO1 variant allele compared to the wild type.
Circumstantial evidence further supporting GSTO1 as having a role in neurodegenerative disorders includes cellular co-localization with IL-1β, which is over-expressed in the brains of both AD and PD patients (Griffin & Mrak, 2002; Czlonkowska et al., 2002) and is a fundamental component of the inflammatory response that is proposed to contribute to the pathogenesis of both AD and PD. Chronopoulou & Labrou (2009) have hypothesised that it is the dehydroascorbate reductase role of GSTO enzymes in the brain which is the basis of their genetic link to AAO in AD and PD.
It is evident from the primary literature that further research as to the role of WT and mut GSTO1 in disease is desirable and an analytical method which facilitates this is required. Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in humans and are associated with differences in disease risk, susceptibility, progression and success of treatment. Genotyping of SNPs is important in disease diagnosis and prognosis and is a key driving force in the expanding sector of personalized medicine. Genotyping techniques which are underpinned by the polymerase chain reaction (PCR) are costly and time-consuming and only enable a ‘risk analysis’ approach to disease diagnostics. In vitro protein detection includes techniques based on electrophoresis, mass spectrometry and antibodies, but each has potential weaknesses with respect to the current problem of wtGSTO1 and mutGSTO1 protein discrimination, in which the structural difference is a single amino acid (out of the 241 of the full protein). For example, electrophoresis is likely to be insufficiently sensitive, mass spectrometry is unlikely to produce distinctive fragmentation patterns, and antibodies to either wild type or mutant are likely to cross-react.
The inventors describe herein an antibody with surprising specificity for wtGSTO1.
References
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